1. Field of the Invention
The present invention relates generally to the field of gene therapy. More particularly, it concerns methods and compositions for increasing transgene expression.
2. Description of Related Art
Gene therapy now is thought to be widely applicable in the treatment of a variety of cancers and a number of other diseases. Viral vectors are one method employed as a gene delivery system. A great variety of viral expression systems have been developed and assessed for their ability to transfer genes into somatic cells. In particular, retroviral and adenovirus based vector systems have been investigated extensively over a decade. Recently, adeno-associated virus (AAV) has emerged as a potential alternative to the more commonly used retroviral and adenoviral vectors. Lipid vectors including cationic lipids and liposomes also are used to deliver plasmid DNA containing therapeutic genes.
The therapeutic treatment of diseases and disorders by gene therapy involves the transfer and stable or transient insertion of new genetic information into cells. The correction of a genetic defect by re-introduction of the normal allele of a gene encoding the desired function has demonstrated that this concept is clinically feasible (Rosenberg et al., New Eng. J. Med., 323:570 (1990)). Indeed, preclinical and clinical studies covering a large range of genetic disorders currently are underway to solve basic issues dealing with gene transfer efficiency, regulation of gene expression, and potential risks of the use of viral vectors. The majority of clinical gene transfer trials that employ viral vectors perform ex vivo gene transfer into target cells which are then administered in vivo. Viral vectors also may be given in vivo but repeated administration may induce neutralizing antibody.
A major issue facing potential clinical application of gene therapy is the question of how to heterologous genes expressed in clinically significant quantities in selected tissues of the subject. Gene regulatory elements provide a potential answer to that question. Gene regulatory elements such as promoters and enhancers possess cell type specific activities and can be activated by certain induction factors via responsive elements. The use of such regulatory elements as promoters to drive gene expression facilitates controlled and restricted expression of heterologous genes in vector constructs. For instance, heat shock promoters can be used to drive expression of a heterologous gene following heat shock.
U.S. Pat. Nos. 5,614,381, 5,646,010 and WO 89/00603, refer to driving transgene expression using heat shock at temperatures greater than 42xc2x0 C. These temperatures are not practicable in human therapy as they can not be maintained for a sustained period of time without harm to the individual.
Gene therapy could be used in combination with a variety of conventional cancer therapy treatments including cytotoxic drugs an radiation therapies. It has been shown that hyperthermia enhances the cell killing effect of radiation in vitro (Harisiadis et al., Cancer, 41:2131-2142 (1978)), significantly enhances tumor response in animal tumors in vivo and improves the outcome in randomized clinical trials. However, the major problem with the use of hyperthermia treatment is that the hyperthermia system can not adequately heat large and deep tumors.
Thus, it would be useful to develop vectors that may be used at temperatures of 42xc2x0 C. and below, systemically or locally, to treat a patient such that the expression of the therapeutic gene(s) is activated preferentially in regions of the body that have been subjected to conditions which induce such expression.
The present invention provides methods for effecting the inducible expression of polynucleotides in cells. In particular, the use of heat shock promoters in methods for effecting the inducible expression of polynucleotides in mammalian cells is taught. The present invention overcomes deficiencies in the prior art by providing heat shock-controlled vectors that may be used at temperatures of 42xc2x0 C. and below. These methods may be used to treat a patient via the inducible expression of a therapeutic gene.
In one embodiment, the present invention provides a method for effecting transgene expression in a mammalian cell that comprises first providing an expression construct that comprises both (i) an inducible promoter operably linked to a gene encoding a transactivating factor and (ii) a second promoter operably linked to a selected polynucleotide. The second promoter is activated by the transactivating factor expressed by the same construct. The method then includes the step of introducing the expression construct into the cell. Finally, the cell is subjected to conditions which activate the inducible promoter and result in the expression of the selected polynucleotide.
In a preferred embodiment of the invention, the inducible promoter is a heat shock promoter and the conditions which activate the heat shock promoter are hyperthermic conditions. The hyperthermic conditions may comprise a temperature between about basal temperature and about 42xc2x0 C. As used herein the basal temperature of the cell is defined as the temperature at which the cell is normally found in its natural state, for example, a cell in skin of a mammal may be at temperatures as low as 33xc2x0 C. whereas a cell in the liver of an organism may be as high as 39xc2x0 C. In specific embodiments, the application of hyperthermia involves raising the temperature of the cell from basal temperature, most typically 37xc2x0 C. to about 42xc2x0 C. or less. Alternatively, the hyperthermic conditions may range from about 38xc2x0 C. to about 41xc2x0 C., or from about 39xc2x0 C. to about 40xc2x0 C. The heat shock promoter is optionally derived from a promoter selected from the group of the heat shock protein (HSP) promoters HSP70, HSP90, HSP60, HSP27, HSP72, HSP73, HSP25 and HSP28. The ubiquitin promoter may also be used as the heat-shock inducible promoter in the expression construct. A minimal heat shock promoter derived from HSP70 an comprising the first approximately 400 bp of the HSP70B promoter may optionally be used in the invention.
In an alternative embodiment, the inducible promoter comprises a hypoxia-responsive element (HRE). This hypoxia-response element may optionally contain at least one binding site for hypoxia-inducible factor-1 (HIF-1).
In one embodiment of the invention, the second promoter may be selected from the group consisting of an human immunodeficiency virus-1 (HIV-1) promoter and a human immunodeficiency virus-2 (HIV-2) promoter. In preferred embodiments, the transactivating factor may be a transactivator of transcription (TAT).
The selected polynucleotide may code for a protein or a polypeptide. For instance, the selected polynucleotide may encode any one of the following proteins: ornithine decarboxylase antizyme protein, p53, p16 , neu, interleukin-1 (IL1), interleukin-2 (IL2), interleukin-4 (IL4), interleukin-7 (IL7), interleukin-12 (IL12), interleukin-15 (IL15), FLT-3 ligand, granulocyte-macrophage stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), gamma-interferon (INFxcex3), alpha-interferon (IFNxcex1), tumor necrosis factor (TNF), herpes simplex virus thymidine kinase (HSV-TK), I-CAM1, human leukocyte antigen-B7 (HLA-B7), or tissue inhibitor of metalloproteinases (TIMP-3). In such an embodiment, the selected polynucleotide is positioned in a sense orientation with respect to the second promoter.
Alternatively, expression of the selected polynucleotide may involve transcription but not translation and produces a ribozyme. In this embodiment, the selected polynucleotide is also positioned in a sense orientation with respect to the second promoter.
In still another alternative embodiment, the expression of the selected polynucleotide involves transcription but not translation and results in an RNA molecule which serves as an antisense nucleic acid. In such an embodiment, the selected polynucleotide may be the target gene, or a fragment thereof, which is positioned in the expression construct in an antisense orientation with respect to said second promoter.
The expression construct may further comprise a gene encoding a selectable marker, such as hygromycin resistance, neomycin resistance, puromycin resistance, zeocin, gpt, DHFR, green fluorescent protein or histadinol. Alternatively, the expression construct may further comprise (i) a second selected polynucleotide which is operably linked to said second promoter, and (ii) an internal ribosome entry site positioned between said first and second selected polynucleotides.
The cell may be a tumor cell, a cell located within a tumor, or a cell located within a mammal. The introduction of the expression construct into the cell may occur in vitro or in vivo. In an one embodiment, the introduction of the expression construct into the cell is mediated by a delivery vehicle selected from the group consisting of liposomes, retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, herpes simplex viruses, and vaccinia viruses.
In another embodiment of the invention, a method of providing a subject with a therapeutically effective amount of a product of a selected gene is provided. This method involves providing a first expression construct which comprises an inducible promoter operably linked to a gene encoding a transactivating factor and providing a second expression construct which comprises a second promoter operably linked to a selected polynucleotide, where the second promoter is activated by the transactivating factor encoded by the first expression construct. The first and second expression construct are introduced into the desired cell of said subject and that cell is subjected to conditions which activate the inducible promoter, so that expression of the selected polynucleotide is induced. In a preferred embodiment, the first and second expression constructs are present on the same vector. Also, the inducible promoter is preferably a heat shock promoter and the activating conditions comprise a temperature below 42xc2x0 C. and above about basal temperature.
The introduction of one or both of the expression constructs may be performed either in vivo or ex vivo. The expression product of the selected polynucleotide may optionally be deleterious to a pathogen in the subject, such as a virus, bacterium, fungus, or parasite. Alteratively, the expression product of the selected polynucleotide may inhibit the growth of the cell of the subject. In still another alternative embodiment of the invention, the expression product of the selected polynucleotide replaces a deficient protein in the subject. Alternatively, the expression product of the selected polynucleotide may promote nerve regeneration.
In further embodiments, there is provided a method of treating cancer in a mammal, such as a human, comprising the steps of (a) providing an expression construct that comprises (i) an inducible promoter, preferably a heat shock promoter, which is operably linked to a gene encoding a transactivating factor; and (ii) a second promoter operably linked to a selected polynucleotide, wherein the second promoter is activated by the transactivating factor; (b) introducing said expression construct into a tumor cell; and (c) subjecting the tumor cell to conditions which activate the inducible promoter so that the selected polynucleotide is expressed in high enough quantities to inhibit the growth of the tumor cell. If the inducible promoter is a heat shock promoter, the activating conditions comprise a temperature below about 42xc2x0 C. and above about basal temperature.
This method further may comprise treating said tumor cell with an established form of therapy for cancer which is selected from the group consisting of external beam radiation therapy, brachytherapy, chemotherapy, and surgery. The cancer may optionally be selected from the group consisting of cancers of the brain, lung, liver, spleen, kidney, lymph node, small intensive, pancreas, blood cells, colon, stomach, breast, endometrium, prostate, testicle, ovary, vulva, cervix, skin, head and neck, esophagus, bone marrow and blood.
In one particular embodiment of the invention, the selected polynucleotide is ornithine decarboxylase antizyme protein. After the cell is subjected to conditions which activate the inducible promoter of the expression construct in the tumor cell, the tumor cell is treated with the radioprotector WR-33278 or WR-1065. Lastly, the tumor cell is treated with radiation therapy.
Methods for provoking an immune response in a mammal, such as a human, are also provided by the present invention. The provoked immune response may constitute either a humoral immune response or a cellular immune response. In one embodiment, the method comprises (a) providing an expression construct that comprises (i) an inducible promoter, preferably a heat shock promoter, which is operably linked to a gene encoding a transactivating factor; and (ii) a second promoter operably linked to a selected polynucleotide, wherein the second promoter is activated by the transactivating factor; (b) introducing said expression construct into a cell in the mammal; and (c) subjecting the cell to conditions which activate the inducible promoter so that the selected polynucleotide is expressed highly enough to provoke an immune response in the mammal. If the inducible promoter is a heat shock promoter, the activating conditions comprise a temperature below about 42xc2x0 C. and above about basal temperature.
In one embodiment, the immune response which is provoked is directed against the cell in the mammal which contains the expression construct. The method may also optionally involve treating the cell with an established form of therapy for cancer selected from the group consisting of chemotherapy, external beam radiation therapy, brachytherapy, and surgery.
In another embodiment, there is provided an expression construct comprising (a) a gene encoding a transactivating factor; (b) an inducible promoter operably linked to the gene; (c) a selected polynucleotide; and (d) a second promoter which is operably linked to the selected polynucleotide. The second promoter of the construct is activated by the transactivating factor. In a preferred embodiment, the inducible promoter is a heat shock promoter and the expression of the selected polynucleotide can be induced by hyperthermic conditions comprising a temperature below about 42xc2x0 C. and above about 37xc2x0 C. In an alternative embodiment, the inducible promoter of the expression construct may comprise a hypoxia-responsive element. The expression construct may also comprise a second selected polynucleotide which is also operably linked to the second promoter and separated by the first selected polynucleotide by a IRES.
A cell comprising the expression construct is also provided. The provided expression construct can also optionally be used in a method of altering the genetic material of a mammal.